The ability of certain factors produced in very low concentration in a variety of tissues to stimulate the growth and development of bone marrow progenitor cells into granulocytes and/or macrophages has been known for nearly 15 years. The presence of such factors in sera, urine samples, and tissue extracts from a number of species is demonstrable using an in vitro assay which measures the stimulation of colony formation by bone marrow cells plated in semi-solid culture medium. There is no known in vivo assay. Because these factors induce the formation of such colonies, the factors collectively have been called Colony Stimulating Factors (CSF).
More recently, it has been shown that there are at least four subclasses of human CSF proteins which can be defined according to the types of cells found in the resultant colonies. One subclass, CSF-1, results in colonies containing macrophages predominantly. Other subclasses produce colonies which contain both neutrophilic granulocytes and macrophages; which contain predominantly neutrophilic granulocytes; and which contain neutrophilic and eosinophilic granulocytes and macrophages.
There are murine factors analogous to the first three of the above human CSFs. In addition, a murine factor called IL-3 induces colonies from murine bone marrow cells which contain all these cell types plus megakaryocytes, erythrocytes, and mast cells, in various combinations. These CSFs have been reviewed by Dexter, T. M., Nature (1984) 309:746, and Vadas, M. A., et al, J Immunol (1983) 130:793.
The invention herein is concerned with the recombinant production of proteins which are members of the first of these subclasses, CSF-1. This subclass has been further characterized and delineated by specific radioimmunoassays and radioreceptor assays--e.g., antibodies raised against purified CSF-1 are able to suppress specifically CSF-1 activity, without affecting the biological activities of the other subclasses, and macrophage cell line J774 contains receptors which bind CSF-1 specifically. A description of these assays was published by Das, S. K., et al, Blood (1981) 58:630.
Purification methods for various CSF proteins have been published and are described in the following paragraphs.
Stanley, E. R., et al, J Biol Chem (1977) 252:4305 reported purification of a CSF protein from murine L929 cells to a specific activity of about 1.times.10.sup.8 units/mg, which also stimulated mainly macrophage production. Waheed, A., et al, Blood (1982) 60:238, described the purification of mouse L-cell CSF-1 to apparent homogeneity using a rabbit antibody column and reported the first 25 amino acids of the murine sequence (Ben-Avram, C. M., et al, Proc Natl Acad Sci (USA) (1985) 882:4486).
Stanley, E. R., et al, J Biol Chem (1977) 252:4305-4312 disclosed a purification procedure for CSF-1 from human urine and Das, S. K., et al, Blood (1981) 58:630; J Biol Chem (1982) 257:13679 obtained a human urinary CSF-1 at a specific activity of 5.times.10.sup.7 units/mg which produced only macrophage colonies, and outlined the relationship of glycosylation of the CSF-1 proteins prepared from cultured mouse L-cells and from human urine to their activities. Wang, F. F., et al, J Cell Biochem (1983) 21:263, isolated human urinary CSF-1 to specific activity of 10.sup.8 U/mg. Waheed, A., et al, disclosed purification of human urinary CSF-1 to a specific activity of 0.7-2.3.times.10.sup.7 U/mg on a rabbit antibody column (Exp Hemat (1984) 12:434).
Wu, M., et al, J Biol Chem (1979) 254:6226 reported the preparation of a CSF protein from cultured human pancreatic carcinoma (MIAPaCa) cells which resulted in the growth of murine granulocytic and macrophagic colonies. The resulting protein had a specific activity of approximately 7.times.10.sup.7 units/rag.
Partially purified preparations of various CSFs have also been reported from human and mouse lung-cell conditioned media (Fojo, S. S., et al, Biochemistry (1978) 17:3109; Burgess, A. W., et al, J Biol Chem (1977) 252:1998); from human T-lymphoblast cells (Lusis, A. J., et al, Blood (1981) 57:13: U.S. Pat. No. 4,438,032); from human placental conditioned medium to apparent homogeneity and specific activity of 7.times.10.sup.7 U/mg (Wu, M., et al, Biochemistry (1980) 19:3846).
A significant difficulty in putting CSF proteins in general, and CSF-1 in particular, to any useful function has been their unavailability in distinct and characterizable form in sufficient amounts to make their employment in therapeutic use practical or even possible. The present invention remedies these deficiencies by providing purified human and murine CSF-1 in useful amounts through recombinant techniques.
A CSF protein of a different subclass, murine and human GM-CSF has been purified and the cDNAs cloned. This protein was shown to be distinct from other CSFs, e.g., CSF-1, by Gough, et al, Nature (1984) 309:763-767. Murine IL-3 has been cloned by Fung, M. C., et al, Nature (1984) 307:233. See also Yokota, T., et al, PNAS (1984) 81:1070-1074; Wong, G. G., et al, Science (1985) 228:810-815; Lee, F., et al, PNAS (1985) 82:4360-4364; and Cantrell, M. A., et al, PNAS (1985) 82:6250-6254.